Anti-cancer and anti-angiogenic effects of curcumin and tetrahydrocurcumin on implanted hepatocellular carcinoma in nude mice

AIM： To determine the effect of tetrahydrocurcumin （THC） on tumor angiogenesis compared with curcumin （CUR） by using both in vitro and in vivo models of human hepatocellular carcinoma cell line （HepG2）. METHODS： The 3-（4,5-dimethylthiazol-2-yl）-2, 5-diphenyl-tetrazolium bromide （MTT） assay was used for testing the anti-proliferating activities of CUR and THC. In male BALB/c nude mice, 2 ×10^6 human HepG2 cells were inoculated onto a dorsal skin-fold chamber. One day after HepG2 inoculation, the experimental groups were fed oral daily with CUR or THC （300 mg/kg or 3000 mg/kg）. On d 7, 14 and 21, the tumor microvasculature was observed using fluorescence videomicroscopy and capillary vascularity （CV） was measured. RESULTS： Pathological angiogenic features including microvascular dilatation, tortuosity, and hyper-permeability were observed. CUR and THC could attenuate these pathologic features. In HepG2-groups, the CV were significantly increased on d 7 （52.43%）, 14 （69.17%）, and 21 （74.08%）, as compared to controls （33.04%,P 〈 0.001）. Treatment with CUR and THC resulted in significant decrease in the CV （P 〈 0.005 and P 〈 0.001, respectively）. In particular, the anti-angiogenic effects of CUR and THC were dose-dependent manner. However, the beneficial effect of THC treatment than CUR was observed, in particular, from the 21 d CV （44.96% and 52.86%, P 〈 0.05）.CONCLUSION： THC expressed its anti-angiogenesis without any cytotoxic activities to HepG2 cells even at the highest doses. It is suggested that anti-angiogenic properties of CUR and THC represent a common potential mechanism for their anti-cancer actions.     

Molecular cloning and characterization of alboaggregin D, a novel platelet activating protein, from Green pit viper (Cryptelytrops albolabris) venom

Viper venoms are abundant sources of proteins affecting hemostasis. This study aimed to clone and purify a high-molecular-weight C-type lectin-like protein (snaclec) from Green pit viper (Cryptelytrops albolabris) venom, as well as to characterize its effects on human platelets.Based on the partial sequences from the C. albolabris venom gland library, we cloned full-length cDNAs encoding the snaclec subunits using 5′RACE and 3′RACE methods. The cDNA sequence of the α subunit contained 477 base pairs (bp) that were translated into 23 amino acid residue signal peptide and a 135-residue mature protein. The cDNA sequence of the β subunit contained 447 bp that were translated into 23-residue signal peptide and a 125-residue mature protein. Compared with known sequences of dimeric snaclecs, these peptides contained extra cysteines that probably formed a high-order multimer. In parallel, a snaclec was isolated from C. albolabris crude venom using gel filtration followed by ion-exchange chromatography. The purified C. albolabris snaclec on SDS-PAGE showed the apparent molecular mass of 120 kDa under native condition and 2 bands of 14 and 17 kD under reduced condition suggesting a tetramer of heterodimers (αβ)₄. Liquid chromatography-tandem mass spectrometry analysis of the peptides found perfect matches with the conceptually translated sequences from the cDNA library. This protein was unique from any other snaclecs previously purified from C. albolabris and named alboaggregin D. It induced human platelet aggregation in the absence of any cofactor with the EC₅₀ of 0.25 nM and caused tyrosine phosphorylation in human platelets. Antibodies against either platelet glycoprotein (GP) Ib or GPVI could inhibit alboaggregin D-induced platelet aggregation. This snaclec may be useful for dissecting the mechanisms of platelet activation.     

Tumor neocapillary density in hepatocellular carcinoma cells implanted nude mice model

Tumor angiogenesis is an important process for several kinds of tumor, especially, during its angiogenic switch. The present study was aimed to investigate the dynamical process of tumor neocapillarization in Hepatocellular carcinoma cells implanted nude mice model. Male BALB/c nude mice (20-25 g) were used. After the implantation of a dorsal skin-fold chamber, the Hepatocellular carcinoma cells (HepG2) (30 microl of 2 x 10(6) cells) were inoculated into the upper layer of the skin within the chamber (HepG2-group), while the shammed control group (Sham-group) was received a normal saline. Intravital fluorescence videomicroscopy was performed to monitor the tumor neocapillary on days 7 and 14 post-inoculation by intravenous injection of 0.1 ml rhodamine B isothiocyanate-labeled dextran (0.5%). Based on the recorded videoimages, the tumor microvascular networks were measured using a digital image analysis. The neovascular density and configuration were represented in terms of microvascular density index (MVDI) and neovascular bifurcation ratio (BR). The MVDI levels of HepG2-group were significantly increased on day 7 and 14 compared to the age-matched Sham-group. The increase in MVDI agreed with the increase in the BR. In conclusion, our dorsal skin-fold chamber technique with intravital fluorescence videomicroscopy and digital image analysis was most useful to monitor the neovascular network for quantitatively evaluating the progression of tumor angiogenesis in terms of MVDI and BR values.     

Development of influenza vaccine production capacity by the Government Pharmaceutical Organization of Thailand: addressing the threat of an influenza pandemic

In 2005, a year after highly pathogenic avian influenza outbreaks in Thailand, the Thai Government issued a National Strategy Plan for Pandemic Influenza Preparedness, a major objective of which was the domestic production of seasonal influenza vaccine. It was considered that sustained influenza vaccine production was the best guarantee of a pandemic vaccine in the event of a future pandemic. The Government decided to provide funds to establish an industrial-scale influenza vaccine production plant, and gave responsibility for this challenging project to the Government Pharmaceutical Organization (GPO). In 2007, with support from the World Health Organization (WHO), the GPO started to develop egg-based, trivalent inactivated influenza vaccine (IIV) in a renovated pilot plant. In early 2009, during the second year of the project, the GPO turned its attention to develop a pandemic live attenuated influenza vaccine (PLAIV) against the influenza A (H1N1) virus. By December 2010, the H1N1 PLAIV had successfully completed Phase II clinical trials and was awaiting registration approval from the Thai Food and Drug Administration (TFDA). The GPO has also started to develop an H5N2 PLAIV, which is expected to enter clinical trials in January 2011. The next step in 2011 will be the development and clinical evaluation of seasonal LAIV. To meet the needs of the national seasonal influenza vaccination programme, the GPO aims to produce 2 million doses of trivalent IIV in 2012 and progressively increase production to the maximum annual capacity of 10 million doses. This article relates how influenza vaccine production capacity was developed and how major challenges are being met in an expeditious manner, with strong local and global commitment.     

Effects of curcumin on tumor angiogenesis and biomarkers, COX-2 and VEGF, in hepatocellular carcinoma cell-implanted nude mice

Anti-angiogenic activity of curcumin and effects of curcumin on angiogenic biomarkers, cycloxygenase (COX)-2 and vascular endothelial growth factor (VEGF) levels were investigated. One day after hepatocellular carcinoma cell (HepG2) cells (30 microl of 2 x 10(6) cells) were inoculated onto the upper layer of the skin-fold chamber (HepG2-group, n = 15), curcumin solutions of 300 and 3000 mg/kg BW were daily oral fed to HepG2-Cur-300 and HepG2-Cur-3000 groups (n = 30), respectively. Intravital fluorescence videomicroscopy was performed to monitor neocapillaries in the tumor on days 3, 7 and 14 post-tumor-inoculation, using RITC-dextran (0.1 ml of 0.5% injected intravenously). The tumor neocapillary density (NCD) was evaluated in correlation with the tumor area, using a digital image analysis. The results demonstrated that the NCD of HepG2-groups were significantly increased on day 7 and 14, compared to the aged-matched Sham-groups (p<0.001). The increased NCD on day 7 and 14 were attenuated significantly by daily treatment of curcumin solution (3000 mg/kg BW).The curcumin treatment reduced the tumor-induced over-expression of COX-2 and serum VEGF in HepG2 groups significantly (p<0.001), indicating that curcumin could inhibit tumor angiogenesis. This mechanism might be mediated through reduction of angiogenic biomarkers, COX-2 and VEGF.     

Antiangiogenic activity of curcumin in hepatocellular carcinoma cells implanted nude mice

Antiangiogenic activity of curcumin on the tumor neogenesis was investigated by evaluating the density of neocapillaries induced by Hepatocellular carcinoma cells (HepG2) in mice, using intravital fluorescence videomicroscopy. Male BALB/c nude mice (20-25 g) were used, and a dorsal skin-fold chamber was implanted. HepG2 (30 microl of 2 x 10(6) cells) were inoculated on the upper surface of the skin within the chamber. The mice were divided into two groups as follows. Dimethyl sulfoxide solution (0.1%) was fed (HepG2 group, n=5) or curcumin solution (3000 mg/kg bw) was fed oral daily (HepG2-Cur group, n=5), one day after the inoculation of HepG. On days 7 and 14 post-tumor-inoculation, the tumor microvasculature was visualized by injecting 0.1 ml of 0.5% rhodamine B isothiocyanate-labeled dextran intravenously, and observed under an intravital fluorescence videomicroscope. Based on the recorded videoimage, the tumor neocapillary density and microvasculature were evaluated using a digital image analysis and correlated with the tumor area. The image analysis demonstrated that in the HepG2-group the neocapillary densities were significantly increased on day 7, and day 14, compared to the aged-matched Sham-group (P<0.05). In the HepG2-Cur group, the increase of tumor neocapillary density was attenuated significantly. It was suggested that high dose of curcumin might be an effective anti-angiogenic drug in the treatment against tumor.